Interferon-β complex

ABSTRACT

The present invention relates to a complex between interferon-β and polyethylene glycol, which has high biological activity, and to a method for producing the complex at high efficiency. Namely, the present invention relates to a method for producing an interferon-β complex comprising binding interferon-β to polyethylene glycol in the presence of at least one additive selected from the group consisting of oligosaccharides having 5 or less sugar units, monosaccharides, their corresponding sugar alcohols, and C 2-6  polyhydric alcohols, and to an interferon-β complex produced by the method, which has polyethylene glycol specifically bound with lysine located at the 19th or 134th position in the amino acid sequence of interferon-β.

TECHNICAL FIELD

The present invention relates to an interferon-β complex havingpolyethylene glycol specifically bound to lysine located at the 19th or134th position in the amino acid sequence of interferon-β, and to aproduction method thereof.

BACKGROUND ART

Water-soluble polymers such as polyethylene glycol, when bound tobiomolecules as typified by protein drugs, are known to confer clinicalusefulness in ways that bring about effects such as improved physicaland thermal stability, resistance to protease, and solubility as well asdecreased in vivo distribution volume and improved retention in blood(see Inada et al., J. Bioact and Compatible Polymers 5,343(1990);Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems9,249 (1992); and Katre, Advanced Drug Delivery Reviews 10,91(1993)).

A variety of methods are available for binding natural interferon-β orinterferon-β having a primary structure identical to natural one to awater-soluble polymer polyethylene glycol (PEG). For example, Katre etal have applied the amino group modification of lysine or the like tothe PEGylation of interferon-β (see U.S. Pat. Nos. 4,766,106 and4,917,888 and International Publication No. WO87/00056). Specifically,they have reported a conjugate obtained by binding a water-solublepolymer (PEG) having a molecular weight of 300 to 100,000 to recombinantinterferon-β or IL-2 via 1 to 10 lysine residues in the amino acidsequence thereof. Alternatively, a technique for binding PEG to an aminogroup in lymphokine has already been reported in “Chemically modifiedlymphokine and production thereof” (see JP Patent Publication (Kokai)No. 60-226821A (1985)). However, in reality, interferon-β bound with PEGby these methods has interferon-β activity decreased to less than 10%and can not be in practical use.

No previous report has described a technique for selectively binding PEGto the amino group of particular lysine in interferon-β. If it ispossible to select and specifically modify lysine that minimizes therate of reduction in interferon-β biological activity caused by PEGbinding, reduction in the total amount of proteins administered as apharmaceutical drug leads to fewer side effects to patients and furtherto easier quality control.

On the other hand, a method is also known which uses reductivealkylation without involving lysine residues to selectively bind awater-soluble polymer to the amino terminus of interferon throughreaction at pH suitable for the selective activation of theamino-terminal α-amino group of the interferon (see JP PatentPublication (Kokai) No. 9-25298A (1997)). However, in reality, thePEGylation of interferon-β by this method does not give mono-PEGylationand brings about nonselective PEGylation at any lysine residue or the Nterminus, resulting in the generation of a heterogeneous mixture withoutsufficient antiviral activity and cell growth-inhibiting activity.

More importantly, purified interferon-β N-terminally bound with PEG isalso known to have remaining activity (ratio with respect tointerferon-β activity before binding) dramatically decreased when thePEG has a molecular weight higher than 20,000 and to completely lackactivity when the PEG has a molecular weight of 40,000, as reported byPepinsky et al (see Pepinsky et al., The Journal of Pharmacology andExperimental Therapeutics, vol. 297, p 1059-1066, (2001)).

As for interferon-α, Bailon et al have produced interferon-αnonselectively mono-PEGylated at the lysine residue with a branchedpolymer PEG having a molecular weight of 40,000 (Bailon et al.,Bioconjugate Chem. 12, 195 (2001)). However, they have reported that theremaining activity of interferon-α bound with PEG having a molecularweight as high as 40,000 is significantly decreased, as in the case withinterferon-β N-terminally bound with PEG, and is 7%.

Namely, it is difficult to directly apply techniques (the number andposition of PEG bound) that have been developed for modification withlow molecular weight PEG to high molecular weight PEG. Thus, a noveltechnique has been required for producing a highly active interferon-βcomplex bound with PEG having a molecular weight (20,000 or higher)necessary to sufficiently obtain effects such as extended in vivocirculatory half-life and decreased clearance values that lead tousefulness as a pharmaceutical drug.

As described above, there has been no report so far on the selection ofa lysine residue to be modified for avoiding reduction in the activityof interferon-β bound with a high molecular weight water-solublepolymer, and on a technique for this purpose. Moreover, there has beenno report that the selective binding of a high molecular weightwater-soluble polymer such as PEG to any one of 11 lysine residuespresent in interferon-β produces a highly active interferon-β complex.

DISCLOSURE OF THE INVENTION

An object of the present invention is to find a structure of aninterferon-β complex that has no impairment of biological activity evenby the modification with a high molecular weight substance such aspolyethylene glycol and to provide a method for producing such complexat high efficiency. Particularly, an object of the present invention isto obtain an interferon-β complex in which 10% or higher of interferon-βactivity is maintained even by the binding of PEG having a molecularweight as high as 40,000.

The present inventors have conducted a variety of studies for attainingthe objects and have consequently found that natural interferon-β hassugar chain-linked asparagine at the 80th position, and means forminimizing reduction in its activity is the selective modification of alysine residue located at 19th or 134th position, which is proximal tothis asparagine from the viewpoint of the three-dimensional structure,with a high molecular weight substance. Even when a polymer (e.g., PEG)having a molecular weight that exceeds 10,000 is used, the selectivebinding of the polymer to the lysine located at the 19th or 134thposition can minimize reduction in interferon-β activity.

The lysine residue located at the 19th position has previously beenlisted as one of lysine residues that should be removed when PEG isbound to the amino group of interferon-β (see International PublicationNo. WO01/15736). WO01/15736 teaches that commercial preparations ofinterferon-β are sold under the name BETASERON® (also termed interferonβ1b, which is non-glycosylated, produced using recombinant bacterialcells, has a deletion of the N-terminal methionine residue and the C17Smutation). Therefore, this binding site cannot be expected fromconventional techniques. As for the lysine residue located at the 134thposition as well, there has been no report so far that the selectivebinding of a high molecular weight modifying substance to this siteminimizes reduction in interferon-β activity.

Namely, the present invention provides a method for producing aninterferon-β complex comprising binding interferon-β to polyethyleneglycol in the presence of at least one additive selected from the groupconsisting of oligosaccharides having 5 or less sugar units,monosaccharides, their corresponding sugar alcohols, and C₂₋₆ polyhydricalcohols. The present invention also provides an interferon-β complexproduced by the method, particularly an interferon-β complexcharacterized in that the complex is produced by specifically bindingpolyethylene glycol to lysine located at the 19th or 134th position inthe amino acid sequence of interferon-β.

The interferon-β complex of the present invention has high bloodsolubility, interferon-β activity, and physical and biological stabilityand is useful as a pharmaceutical drug in the treatment, prevention, andalleviation of all symptoms and diseases to which interferon-β isapplied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the Poros HS column separation andpurification of an interferon-β complex having polyethylene glycol boundwith the amino group of lysine located at the 19th position. In thedrawing, the upper arrow denotes the proportion of Solvent B mixed, andthe lower arrow denotes absorbance at 280 nm;

FIG. 2 is a diagram showing a result of analyzing components of peaks 1to 4 (in the drawing, (i) to (iv)) obtained by the Poros HS columnseparation and purification of an interferon-β complex havingpolyethylene glycol bound with the amino group of lysine located at the19th position, wherein the components are separated by SDS-PAGE and thenanalyzed by silverstaining;

FIG. 3 is a diagram showing the SP-5PW column separation andpurification of an interferon-β complex having polyethylene glycol boundwith the amino group of lysine located at the 19th position;

FIG. 4 is a diagram showing the amino acid sequence of interferon-β SEQID NO: 1 and predicted lysyl endopeptidase cleavage sites therein;

FIG. 5 is a diagram showing the peptide map (treated with lysylendopeptidase) of eluted components of peaks 2 to 4 (in the drawing,indicated by (ii) to (iv)) separated with SP-5PW column after bindingreaction with PEG having a molecular weight of 40K as well as thepeptide map of interferon-β(in the drawing, indicated by Pre in thelowermost part) before reaction with PEG;

FIG. 6 is a diagram showing the retention, in rabbit blood, ofinterferon-β having 40K-molecular weight PEG bound with lysine locatedat the 19th position;

FIG. 7 is a diagram showing time course of induction of apharmacological marker (2-5A synthetase activity) in a rabbit byinterferon-β having 40K-molecular weight PEG bound to lysine located atthe 19th position;

FIG. 8 is a diagram showing a result of analyzing peak fractionsobtained by the TOYOPEARL CM 650 column separation of an interferon-βcomplex having polyethylene glycol bound with the amino group of lysinelocated at the 134th position, wherein the fractions are analyzed bySDS-PAGE (A), and the fraction of the peak 3 (in the drawing, (iii)) isfurther analyzed with SP-5PW column (B) (each of the chromatograph waslined from bottom to top in the order of elution);

FIG. 9 is a showing the activities of a nonselectivelymultiply-PEGylated interferon-β complex with 2 or more PEG molecules anda mono-PEGylated interferon-β complex with PEG selectively bound withlysine located at the 19th or 134th position. In the drawing, achromatogram obtained with TOYOPEARL CM 650(S) column (Tosoh) (B), aresult of SDS-PAGE analysis corresponding to each separated fraction(A), and an antiviral activity value per amount of proteins of eachfraction and an activity retention rate relative to the specificactivity of IFN-β before PEG binding (C) are shown in correspondencewith one another; and

FIG. 10 is a diagram showing the retention, in blood, of an IFN-βcomplex bound with 20,000 (20K)- or 40,000 (40K)-molecular weight PEG,which has been administered intravenously into a rabbit.

The present specification encompasses contents described in thespecification of Japanese Patent Application No. 2003-299850 that servesas a basis for the priority of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

As described above, a method of the present invention can efficientlybind polyethylene glycol to lysine located at the 19th or 134th positionin the amino acid sequence of interferon-β and separate unbound productsand by-products therefrom.

Natural interferon-β, interferon-β having a sugar chain altered fromnatural one, or recombinant interferon-β with or without a sugar chaincan be used as the interferon-β subjected to the method of the presentinvention. In the method of the present invention, a commerciallyavailable product may be used as such interferon-β. Natural interferon-βhas sugar chain-linked asparagine at the 80th position that is proximalto lysine located at 19th position from the viewpoint of thethree-dimensional structure. When high molecular weight PEG or the likeis bound thereto, it is preferred to use recombinant interferon-βwithout a sugar chain because possible steric hindrance reduces reactionefficiency. Any of those having the amino acid sequence of naturalinterferon-β with the deletion, substitution, or addition of one orseveral amino acids can also be used as the interferon-β subjected tothe method of the present invention.

The interferon-β of the present invention also encompasses naturalinterferon-β, recombinant interferon-β, and an altered form thereof. Thealtered form means any of those obtained by altering or modifying theamino acid sequence or sugar chain of natural interferon as describedabove. In the present specification, the lysine located at the 19th or134th position is represented by the amino acid number for the aminoacid sequence of this natural interferon-β (FIG. 4 and SEQ ID NO: 1).The amino acid number of such lysine for the altered form is changed inways that correspond to the position of the lysine in the amino acidsequence of the natural interferon-β.

The interferon-β in any of these forms may be obtained by any methodsuch as extraction from tissue, protein synthesis, and biologicalproduction using natural or recombinant cells. Genetically engineeredinterferon-β without a sugar chain is commercially available, and suchcommercially available interferon-β can also be used in the method ofthe present invention.

Polyethylene glycol (PEG) is harmless to human bodies and, whenadministered as an interferon-β complex bound therewith, confers watersolubility at a level necessary to dissolve the complex in blood. It isknown in the art that the binding of PEG to a physiologically activesubstance allows the physiologically active substance in living bodiesto attain improved physical and thermal stability, protection againstenzymatic degradation, enhanced solubility, extended in vivo circulatoryhalf-life, and decreased clearance values. In light of such effects, PEGcan preferably be used in the present invention.

Any method may be used in PEG terminal activation for binding PEG to theamino group of the lysine residue in interferon-β. For example, PEGhaving an amino-reactive structure such as a hydroxysuccinimide ester ornitrobenzene sulfonate ester structure at the terminus can be employed.In the present specification, any of these terminal structures isreferred to as an “amino-reactive functional group,” and PEG having anyof these terminal structures is referred to as “polyethylene glycolactivated with an amino-reactive functional group.” The PEG having anyof these structures is conventionally in wide use for the binding withan amino group and can be produced with ease by a synthetic methodcommonly known or is commercially available. In the present invention,such a commercially available product can also preferably be used.

The average molecular weight of the PEG is not particularly limited andhowever, is preferably approximately 10,000 to 60,000, more preferablyapproximately 20,000 to 40,000, from the viewpoint of allowinginterferon-β in living bodies to attain physical and thermal stability,protection against enzymatic degradation, enhanced solubility, extendedin vivo circulatory half-life, and decreased clearance values.

The binding reaction between interferon-β and PEG can be performed byreacting interferon-β with PEG at pH 5.0 to 8.5, preferably pH 5.5 to8.0, and in the presence of an anti-reduction agent for interferon-βactivity, preferably in a buffer solution such as phosphate or citratebuffer solutions. The anti-reduction agent for interferon-β activity notonly suppresses the aggregation of interferon-β caused by its beingplaced under the atmosphere of pH 5.0 to 8.5 suitable for the reaction,but also helps the specific binding reaction of PEG to the targetedlysine located at the 19th or 134th position or a site proximal thereto.Examples of the anti-reduction agent for interferon-β activity forefficiently binding PEG to the desired site with interferon-β activitymaintained can include saccharides, among others, oligosaccharideshaving 5 or less sugar units, monosaccharides, their corresponding sugaralcohols, C₂₋₆ polyhydric alcohols. Particularly preferred aredisaccharides or monosaccharides such as glucose, mannitol, sorbitol,sucrose, or trehalose, their corresponding sugar alcohols, and C₂₋₃polyhydric alcohols such as ethylene glycol or glycerol. Theseanti-reduction agents for interferon-β activity can be used alone or inany combination of two or more of them.

The concentration of the anti-reduction agent for interferon-β activitysubjected to the method of the present invention is not particularlylimited and however, is approximately 1 to 90% (in total, when pluralanti-reduction agents for interferon-β activity are used; hereinafter,specified in the same way), more preferably approximately 1 to 50%, evenmore preferably approximately 10 to 30%, with respect to the totalweight of the reaction mixture. An interferon-β: PEG mixture ratio isnot particularly limited and however, is typically approximately 1:1 to1:400 molar ratio and preferably approximately 1:4 to 1:100 molar ratiofor PEG activated with succinimidyl ester. A reaction temperaturesuitable for the method of the present invention is typically 4 to 40°C., preferably 4 to 25° C. Although a reaction time is appropriatelydetermined according to the reaction temperature and so on,approximately 1 hour to 24 hours are typically adequate.

Polyethylene glycol can be bound specifically to lysine located at the19th or 134th position in the amino acid sequence of interferon-β or toa site sterically proximal thereto by the reaction process. The “sitesterically proximal thereto” refers to a nearby site in the activeconformation of interferon-β and concretely, is cysteine located at the17th position or asparagine located at the 80th position (particularly,a sugar chain linked thereto) for the lysine located at the 19thposition. Moreover, the term “specific” or “specifically” refers to theselective and preferential binding of polyethylene glycol to the lysinelocated at the 19th or 134th position or to the site sterically proximalthereto. This specific binding gives homogeneous mono-PEGylatedinterferon-β.

A water-soluble polymer having a thiol-reactive structure such as anorthopyridyl disulfide, vinyl sulfone, maleimide, or iodoacetamidestructure at the terminus, preferably a water-soluble polymer having amaleimide structure, is used in binding reaction with the thiol group ofcysteine. For binding PEG having a particularly desirable molecularweight of 10,000 to 60,000 to the cysteine residue in the amino acidsequence of interferon-β, it is preferred to use interferon-β having asugar chain smaller than natural one, interferon-β from which a sugarchain has been removed, or interferon-β originally having no sugarchain. The use of such interferon-β allows binding reaction withoutreductive dissociation to proceed a thigh efficiency in a single step.

After binding reaction, unreacted interferon-β and PEG and by-productscan be removed by any of or any combination of methods such aschromatography using an ion exchange carrier, a gel filtration carrier,or a hydrophobic or hydrophilic carrier, to purify or concentrate thedesired interferon-β complex having PEG bound with lysine located at the19th or 134th position.

One of methods to most efficiently purify and concentrate theinterferon-β complex having PEG bound with lysine located at the 19th or134th position is chromatogram using an ion exchange carrier. The ionexchange carrier used is preferably a cation exchange carrier, morepreferably a carrier where a sulfopropyl, sulfonic acid, orcarboxymethyl group is attached to a base material, and any of these ionexchange carriers is commercially available. For example, when HiTrap SPHP (Amersham Pharmacia), Poros HS (Applied Biosystems), or SP-5PW(Tosoh) is used, a di-PEGylated interferon-β complex present in traceamounts in the reaction solution is initially eluted by asalt-concentration gradient. Subsequently, the desired interferon-βcomplex having PEG bound with lysine located at the 19th position in theamino acid sequence of interferon-β is eluted at a proportion of 40% ormore of the total eluted fractions, followed by the elution andfractionation of complexes having PEG bound with N-terminal amino groupor lysine located at the 33rd, 46th, or 108th position as minor isomersof PEG-bound sites, and unreacted interferon-β. In this procedure, theinterferon-β complex having PEG bound with lysine located at the 134thposition can be isolated at the same time.

Binding to the cation exchange carrier is performed by adjusting thereaction solution to ion strength suitable for the binding at pH 3.0 to8.0. In this case, the cation exchange carrier may be loaded onto acolumn or suspended in the reaction solution. However, when theaggregation of an unreacted hydrophilic polymer in the cation exchangecarrier reduces the separation efficiency of the desired complex, it ispreferred to load the carrier onto the column after suspending andbinding to perform elution. Elution from the cation exchange carrier canbe performed by conducting stepwise gradient or isocratic elution withincreasing salt concentrations or pH in a buffer solution composed ofcitrate, acetate, phosphate, or the like.

The PEG-bound site in the fractionated and eluted interferon-β complexcan be analyzed, as described in Example 3, by peptide mapping, followedby the amino acid analysis or sequencing of the obtained PEG-boundfragment.

The antiviral activity of the interferon-β complex having PEG bound withlysine located at the 19th or 134th position thus produced can bemeasured with ease by a method known in the art (e.g., Armstrong, J. A.,Methods In Enzymology, 78, 381-387, (1981); Rubinstein et al., J. Virol.37, 755 (1981); and Borden et. al., Canc. Res. 42, 4948 (1982)).Interferon-β having 40,000-molecular weight PEG bound with lysinelocated at the 19th position maintains 10% or higher of activity beforebinding, and this activity is equivalent to the activity of interferon-βbound with PEG having a molecular weight of 20,000. Alternatively,interferon-β having 40,000-molecular weight PEG bound with lysinelocated at the 134th position maintains 70 to 100% of activity beforebinding. The remaining activity of a complex having 40,000-molecularweight PEG bound with the N terminus of interferon-β has previously beenreported to be 0% (Pepinsky et al., The Journal of Pharmacology andExperimental Therapeutics, vol. 297, p 1059-1066, (2001)). Accordingly,this demonstrates that the use of the lysine located at the 19th or134th position as the high molecular weight PEG-bound site ofinterferon-β is exceedingly useful.

The method of the present invention can also be applied to substancesother than PEG as a method for producing a complex without reduction ininterferon-β activity. Preferably, the substance other than PEG has anamino-reactive structure such as a hydroxysuccinimide ester ornitrobenzene sulfonate ester structure. This second molecule is notlimited to molecules for conferring in vivo stability such as PEG andserum proteins and may be a physiologically active substance havingtotally different function such as enzymes, cytokine, antibodymolecules, or fragments thereof. A complex derived from any of thesesubstances is useful for constructing a fusion molecule or labelingagent also having interferon-β activity.

In addition, the method of the present invention can be applied to theimmobilization of interferon-β onto a variety of supports, for example,the flat surface or granule of a sugar, glass, or resin material.Namely, the use of the lysine located at the 19th or 134th position as abinding point between interferon-β and any of a variety of supportsallows the immobilization of the interferon-β without reduction in itsactivity. This immobilization procedure requires introducing across-linking agent having a similar amino-reactive functional group orbinding the cross-linking agent to the support in advance.

The complex between interferon-β and PEG of the present invention can beused in the treatment of a variety of diseases that exploits IFNbiological activity. For example, the complex can be used in thetreatment of chronic active hepatitis B, chronic hepatitis C, and otherviral diseases; a variety of malignant neoplasms such as glioblastoma,medulloblastoma, astrocytoma, and malignant melanoma of skin; andautoimmune diseases such as multiple sclerosis. Furthermore, it can beused in the treatment of disease accompanying vascularization, forexample, inflammatory disease (e.g., rheumatic arthritis or psoriasis),eye diseases (e.g., diabetic retinopathy, retinopathy of prematurity,neovascular glaucoma, Stevens-Johnson syndrome and its related disease,ocular pemphigoid and its related disease, chemical burn of cornea, ortrachoma), and cancer (e.g., breast cancer, prostatic cancer, malignantmelanoma, renal cancer, brain tumors, or Kaposi's sarcoma).

The interferon-β complex of the present invention can be administeredthrough an oral or parenteral route, either directly or as apharmaceutical composition prepared by mixing the complex with apharmacologically acceptable carrier or excipient known in the art.However, administration performed by hypodermic, intramuscular, orintravenous injection is preferred.

Concrete examples of a dosage form for oral administration includetablets, pills, capsules, granules, syrups, emulsions, and suspensions.Such dosage forms are produced by a method per se known in the art andcontain a carrier or excipient typically used in a pharmaceutical field.

Examples of the carrier or excipient for tablets include lactose,maltose, saccharose, starch, and magnesium stearate. Examples of adosage form for parenteral administration include eye-drops, ointments,injections, poultices, suppositories, transnasal absorption agents,transpulmonary absorption agents, transdermal absorption agents, andlocally sustained-release agents.

Liquid preparations can be prepared by a method known in the art, forexample, by dissolving or suspending the interferon-β complex in asterile aqueous solution typically used for injections or by theemulsification or the embedding into liposome, of the interferon-βcomplex.

Solid preparations can be prepared by a method known in the art, forexample, by adding an excipient such as mannitol, trehalose, sorbitol,lactose, or glucose to the interferon-β complex to make a freeze-driedproduct. This freeze-dried product can further be powdered, orotherwise, this powder can be mixed and solidified with polylactic acidor glycolic acid for use.

Gelling agents can be prepared by a method known in the art, forexample, by dissolving the interferon-β complex in a thickener orpolysaccharide such as glycerin, polyethylene glycol, methylcellulose,carboxymethylcellulose, hyaluronic acid, or chondroitin sulfate. Any ofthese preparations can be supplemented with human serum albumin, humanimmunoglobulin, α2-macroglobulin, amino acid, or the like, as astabilizer and can be supplemented with alcohol, sugar alcohol, an ionicsurfactant, a nonionic surfactant, or the like, as a dispersant orabsorption promoter within a range that does not impair IFN biologicalactivity. Alternatively, trace metal or a salt of an organic acid canoptionally be added thereto.

The dose of the complex of the present invention is appropriatelydetermined according to the age and body weight of a patient, disease orsymptoms to be treated, an administration form and route, the molecularweight of PEG, and so on. However, in general, the complex of thepresent invention is administered within a range of one dose/month toone dose/day, preferably one dose/month to one dose/week, with 1,000units to 100 million units/dose, preferably 10,000 units to 18 millionunits/dose.

EXAMPLES

Hereinafter, the present invention will be described more fully withreference to Examples.

Example 1

Effect of Additive on Binding Reaction of Polyethylene Glycol Activatedwith Hydroxysuccinimide Ester to Amino Group in RecombinantInterferon-β:

Glucose, glycerol, or ethylene glycol was added at each finalconcentration of 1, 5, 10, and 20% to recombinant human interferon-β(final concentration: 200 μg/ml; which was expressed and purified withrecombinant Escherichia coli according to the method of Goeddel et al,Nucleic Acid. Res. Vol. 8, 4057-4074 (1980)) stored in 0.5 M sodiumchloride and 100 mM acetate buffer solution (pH 5.0). The pH of thesesolutions and a control free of the additive was adjusted to 7.8 using 1M disodium hydrogenphosphate solution. Polyethylene glycol (averagemolecular weight: 40K; manufactured by Shearwater Polymers, INC andpurchased from NOF Corp) activated with hydroxysuccinimide ester wasmixed at a molar ratio of approximately 10 per mole of interferon-β witheach of the resulting solutions, followed by binding reaction overnightat 4° C. After reaction, unreacted interferon-β was removed, andinterferon-β activity in each of the prepared reaction solutions wasmeasured.

The measurement of the activity was performed using enzyme antibodytechnique (sandwich immunoassay) (see Eiji Ishikawa, “EnzymeImmunoassay” 3rd Ed., p. 180, Igaku-shoin). Specifically, rabbitanti-interferon-β antibodies were immobilized on an immunoplate, towhich enzyme-labeled mouse monoclonal antibodies that recognized onlyactive interferon-β structures were then added together with the sample.After the washout of unbound products, a color substrate was added tothe immunoplate to calculate the interferon-β activity of the sample bycomparison with the coloring value of a standard (a result oninterferon-β activity was confirmed to be equal to a result obtained bya biological activity measurement method based on the antiviral activityof cultured cells). Meanwhile, the addition of a surfactant Tween 80 orHCO-60 in the same way as above suppressed the aggregation ofinterferon-β and however, also largely suppressed the progression of thebinding reaction of PEG, leading to unsuccessful measurement of theactivity of the conjugate.

As shown in Table 1, an evident effect of improving activity wasobserved in the reaction solution containing a proper amount of glucose,glycerol, or ethylene glycol, as compared with the control (activity ofthe PEG-interferon-β complex obtained by the binding reaction of PEG inthe absence of the additive).

TABLE 1 Interferon-β activity (10E+7 IU) Per reaction Per AdditiveConcentration(%) solution weight of protein 1 Glucose 1 1.79 1.62 2 51.95 2.46 3 10 2.49 4.96 4 20 2.62 5.03 5 Glycerol 1 2.28 1.73 6 5 2.191.87 7 10 2.53 1.81 8 20 2.51 2.24 9 Ethylene glycol 1 1.74 1.37 10 52.24 1.68 11 10 2.77 2.04 12 20 2.96 2.37 13 Absent 1.70 1.09

Example 2

Separation and purification of interferon-β complex having polyethyleneglycol bound with amino group of lysine located at 19th position:

Ethylene glycol was added at the final concentration of 20% torecombinant human interferon-β (final concentration: 200 μg/ml) storedin 0.5 M sodium chloride and 100 mM acetate buffer solution (pH 5.0),followed by pH adjustment to 7.6 using 1M disodium hydrogenphosphatesolution. Polyethylene glycol (average molecular weight: 40K; purchasedfrom NOF Corp) activated with hydroxysuccinimide ester was mixed withthe resulting solution, followed by binding reaction overnight at 4° C.The reaction solution was dialyzed overnight at 4° C. against 20 mMacetate buffer solution (pH 4.5) containing 10 mM NaCl-0.05% Tween 20.The dialyzed solution was applied to a cation exchange column Poros HS1.7 mL-gel (manufactured by Applied Biosystems) or SP-5PW (Tosoh).Elution was performed by increasing the proportion of Solvent B (20 mMacetate buffer solution (pH 4.5 to 4.7) containing 1 M NaCl) mixed toSolvent A (20 mM acetate buffer solution (pH 4.5 to 4.7) containing 10mM NaCl). Specifically, elution was performed by stepwise increasing theproportion of Solvent B to 30, 40, 50, and 100% in the Poros HS columnand by using 0 to 100% continuous gradient in the SP-5PW column. Anabsorbance chromatogram obtained by elution with the Poros HS column isshown in FIG. 1. Components on the absorbance chromatogram eluted byincreasing stepwise the proportion of Solvent B to 30, 40, 50, and 100%are designated as peaks 1 to 4 (in the drawing, (i) to (iv)),respectively.

A result of analyzing each peak component (1 to 4) by silver stainingafter SDS-PAGE separation is shown in FIG. 2. The desired interferon-βcomplex having 40K-molecular weight PEG bound with the lysine residuelocated at the 19th position could be obtained in the peak 2. Minorisomers of PEG-bound sites that could not quite be controlled byreaction could be separated as by-products, which include aninterferon-β complex having PEG bound with a lysine residue located atthe 33rd position (in the peak 3) and an interferon-β complex having PEGbound with an N-terminal amino group or a lysine residue located at the108th or 134th position (in the peak 4). Unreacted interferon-β anddi-PEGylated interferon-β could be separated in the peaks 4 and 1,respectively.

An absorbance chromatogram obtained by elution with the SP-5PW column isshown in FIG. 3. The SP-5PW column was capable of separation similar tothe Poros HS column separation. The desired interferon-β complex having40K-molecular weight PEG bound with the lysine residue located at the19th position accounted for, as the peak 2, approximately 65% of thetotal amount of proteins (its proportion was 65% or more to allPEGylated complexes except unreacted interferon-β).

Example 3

Confirmation of polyethylene glycol-bound site of recombinantinterferon-β:

Each peak fraction separated with the SP-5PW column in Example 2 wasdesalted and concentrated with a solid-phase extraction cartridge (OASISHLB; Waters) and then exsiccated with a centrifuge evaporator. Theresulting product was dissolved in a Tris buffer solution (pH 9)containing 6 mol/L guanidine, followed by Cys reduction withdithiothreitol and carboxyamidomethylation with iodoacetamide. After theaddition of lysyl endopeptidase, the resulting mixture was incubated at37° C. for 5 hours to perform structure-specific digestion. The enzymereaction was terminated with acetic acid to make a pretreated sample foranalysis.

This sample was subjected to reverse-phase HPLC analysis under thefollowing conditions: column: Cadenza CD-C (184.6×150); detectionwavelength: 214 nm (UV); column temperature: 40° C.; flow rate: 0.8mL/min; mobile phase A: acetic acid/TFA/distilled water (1/0.2/1000);mobile phase B: acetic acid/TFA/acetonitrile/distilled water(0.9/0.2/800/200); gradient: 5% to 70% mobile phase B in 80 min,followed by 70% to 100% mobile phase B in 5 min; and analysis cycle: 120min.

Peaks (K1 to K12) in HPLC chromatogram corresponding to lysylendopeptidase digestion fragments of interferon-β before the bindingreaction of PEG are shown in FIGS. 4 and 5-pre. The arrows in FIG. 4denote lysyl endopeptidase cleavage sites. Peptide fragments generatedby cleavage were designated as K1 to K12. The symbols K1 to K12 in FIG.5 correspond to the peptide fragments K1 to K12 in FIG. 4, respectively.In contrast, the remarkable decrease of the peptide fragments K1 and K2was observed in the peptide map of the peak 2, as shown in FIG. 5-2 (inthe drawing, (ii); hereinafter, specified in the same way). This isprobably because the introduction of PEG to the amino group on the sidechain of lysine located at the 19th position allowed this site tocircumvent lysyl endopeptidase digestion, resulting in no generation ofthe peptide fragments K1 and K2. From this result, the site where PEGwas introduced was estimated to be lysine located at the 19th position.

The peptide map of the peak 3 produced a result shown in FIG. 5-3, inwhich the remarkable absent of the peptide fragment K2 was observed.This is probably because the introduction of PEG to the amino group onthe side chain of lysine located at the 33rd position allowed this siteto circumvent lysyl endopeptidase digestion, resulting in no generationof the peptide fragment K2. From this result, the main site where PEGwas introduced was estimated to be Lys 33.

Because a decrease in the peptide fragment K1 was observed in the peak 4as shown in FIG. 5-4, the presence of an N-terminal conjugate wasestimated. In addition, the peptide fragment K10 was decreased one-half,suggesting that a Lys 134 or Lys 123 isomer was likely to be contained.

Next, a PEG-peptide conjugate fragment that appeared as a peak around 75minutes in the reverse-phase HPLC analysis of peaks was subjected toamino acid sequence analysis. This result and information obtained fromthe peptide map demonstrated that the peak 2, a main reaction product,is the desired complex having PEG bound with lysine located at the 19thposition. A positional isomer having PEG bound with lysine located atthe 33rd position and a positional isomer having PEG bound with lysinelocated at the 134th or 108th position or the N terminus were separatedin the peaks 3 and 4, respectively, as minor by-products.

Example 4

Measurement of remaining activity of interferon-β complex having 40K- or20K-molecular weight peg selectively bound with lysine residue locatedat 19th position:

A recombinant human interferon-β complex having 40K- or 20K-molecularweight PEG selectively bound with the lysine residue located at the 19thposition was synthesized, isolated, and purified by the method ofExample 2, followed by activity comparison with recombinant humaninterferon-β before PEG binding. The comparison of interferon-β activitywas made by measuring antiviral activity. Specifically, the assessmentwas made by bioassay using human amniocytes FL cells in combination withsindbis viruses or vesicular stomatitis viruses (VSV) (Armstrong, J. A.,Methods In Enzymology, 78, 381-387, (1981)).

As a result, the activity of recombinant human interferon-β before PEGbinding was 1.22×10⁸ MIU/mg, whereas the conjugate having 40K PEG hadantiviral activity of 5.5×10⁷ MfU/mg and a remaining activity value ashigh as 45%. The remaining activity of the conjugate having 20K PEGmeasured in the same way was 38.7%.

Example 5

Pharmacokinetic analysis of interferon-β complex having 40K-molecularweight PEG selectively bound to lysine residue located at 19th positionand evaluation of its activity of inducing pharmacodynamic marker:

A recombinant human interferon-β complex having 40K-molecular weight PEGselectively bound with the lysine residue located at the 19th positionwas synthesized, isolated, and purified by the method of Example 2. Thisinterferon-β complex was administered at 9 MIU/kg to a rabbit (NZW,male). Blood was collected from the rabbit before administration andafter 15 minutes, 1.5 hours, 3.5 hours, 8 hours, 1 day, 2 days, 3 days,4 days, 5 days, 6 days, and 7 days of administration to measureantiviral activity in plasma and 2-5A synthetase activity in wholeblood. The antiviral activity was measured by the method described inExample 1, while the 2-5AS synthetase activity was measured using 2-5AKit “Eiken” (Eiken Chemical) according to the specified protocol. Thetime course of the remaining activity of interferon-β in blood based onthe antiviral activity measurement is shown in graph form in FIG. 6. Thetime course of 2-5AS synthetase activity serving as a pharmacodynamicmarker is shown in graph form in FIG. 7. The binding of PEG having amolecular weight of 40K resulted in 20.8-fold increase in the remainingactivity (AUC) of interferon-β in blood. This increase led to a rise inthe activity of inducing the pharmacodynamic marker (AUC was increasedby 7.6 times by the binding of PEG and exceeded the highest value of theinduction of the pharmacodynamic marker by unmodified interferon-β evenafter 7 days post administration).

Example 6

Separation and purification of interferon-β complex having polyethyleneglycol bound with amino group of lysine located at 134th position:

A reaction solution of the binding between recombinant humaninterferon-β and PEG obtained in the same way as in Example 2 wassupplemented with a 5-fold volume of 10 mM acetate buffer solution (pH4.5) and applied to a cation exchange column (TOYOPEARL CM 650(S)(Tosoh)) equilibrated with the same buffer solution.

Proteins were eluted with the same buffer solution containing 1 M sodiumchloride by increasing the proportion of the buffer solution mixed from0 to 65% in a continuous gradient, and then fractionated. The elutedfractions were analyzed by SDS-PAGE and with SP-5PW column (Tosoh). Therespective results are shown in FIGS. 8-A and 8-B.

As a result, three peaks were obtained as in Example 2. However, whenthe fraction contained in the third peak (peak (iii) in FIG. 8-A) wasseparately analyzed with the SP-5PW column, the fraction was shown to befurther separated into several components (FIG. 8-B). Among thesecomponents, the fraction containing a peak (arrow in FIG. 8-B) thatconstituted the highest percentage of the third peak and was eluted lastwas analyzed in the same way as in Example 3. As a result, an IFN-βcomplex having PEG bound with lysine located at the 134th position wasisolated therein.

Example 7

Comparison of activity between peg interferon-β complex obtained bynonselective binding reaction of peg to lysine and peg interferon-βcomplex obtained by selective binding reaction thereof:

Ethylene glycol was added at the final concentration of 20% torecombinant human interferon-β or natural interferon stored in 0.5 Msodium chloride and 100 mM acetate buffer solution (pH 5.0), followed bypH adjustment to 5.5 (reaction condition 1) or to approximately 7.6(reaction condition 2) using 1M disodium hydrogenphosphate solution, inthe same way as in Example 2. Polyethylene glycol (average molecularweight: 10K, 20K, or 40K; manufactured by Shearwater Polymers, INC andpurchased from NOF Corp) activated with hydroxysuccinimide ester wasmixed in a 45-fold amount relative to one interferon-β molecule with theresulting solution, followed by binding reaction overnight at 4° C.

At the same time, SDS was added at the final concentration of 0.1% to arecombinant human interferon-β or natural interferon-β solution,followed by the pH adjustment of the reaction solution to 9.0 (reactioncondition 3). Polyethylene glycol (average molecular weight: 10K, 20K,or 40K) activated with hydroxysuccinimide ester was mixed in a 45-foldamount relative to one interferon-β molecule with the resultingsolution, followed by binding reaction overnight at 4° C.

Interferon-β activity in each of the solutions after reaction wasevaluated by the same antiviral activity measurement method as inExample 4. The progression of binding reaction in each of the solutionswas confirmed by SDS-PAGE.

As shown in Table 2, interferon-β activity was decreased to 10% or lessregardless of the molecular weight of PEG under the reaction condition 3that did not secure the binding selectivity of PEG to lysine. On theother hand, at least 10% or higher of interferon-β activity wasconfirmed to be maintained regardless of the molecular weight of PEGunder the reaction conditions 1 and 2 that enhanced the bindingselectivity of PEG to lysine located at the 19th or 134th position.

TABLE 2 IFN-β type Natural (sugar chain-linked) IFN-β E. colirecombinant IFN-β PEG molecular weight 40K 20K 10K 40K 20K 10K Reaction100% 86% 94% 94% 54% 61.9% condition 1 Reaction 54% 29% 15% 48% 21%  22% condition 2 Reaction 1% 2% 1% 2.8%  0.7%   1.5% condition 3

Example 8

Comparison of activity between nonselectively multiply-PEGylatedinterferon-β complex with 2 or more PEG molecules and mono-PEGylatedinterferon-β complex with PEG selectively bound with lysine located at19th or 134th position:

After the binding reaction of PEG in the same way as in Example 6, afraction containing a nonselectively multiply-PEGylated interferon-βcomplex with 2 or more PEG molecules and a fraction containing amono-PEGylated interferon-β complex with PEG selectively bound withlysine located at the 19th or 134th position were separated withTOYOPEARL CM 650(S) column (To soh) to measure their interferon-βactivities by the method of measuring the antiviral activity describedin Example 4. As a result, as shown in FIG. 9, the nonselectivelymultiply-PEGylated interferon-β complex with 2 or more PEG moleculesmaintained only approximately 1% activity, while the mono -PEGylatedinterferon-β complex with 40K PEG selectively bound with lysine locatedat the 19th or 134th position maintained 10% or more activity.

Example 9

Comparison of retention in blood between IFN-β complex bound with20,000-molecular weight PEG and IFN-β complex bound with40,000-molecular weight PEG:

IFN-β bound with 20,000- or 40,000-molecular weight PEG andnon-PEGylated IFN-β were labeled with 125I and intravenouslyadministered to a rabbit. Blood was chronologically collected from therabbit up to 6 days. The amount of each interferon-β remaining in bloodwas measured by measuring radio activity with a γ-counter. Time courseof the amount of interferon-β remaining in blood are shown in FIG. 10,with radio activity at the time of administration as 100%. The integralof the amount of interferon-β remaining in blood for IFN-β bound with40,000-molecular weight PEG up to 6 days gave a rise 5.5 times greaterthan that of non-PEGylated IFN-β. The integral of the amount ofinterferon-β remaining in blood for IFN-β bound with 20,000-molecularweight PEG stayed at a rise 1.5 times greater than that of non-PEGylatedIFN-β. This result demonstrated that it is important for the retentionof the IFN-β complex in blood to bind 20,000 or more molecular weightPEG to IFN-β, with activity maintained.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, polyethylene glycol can be boundspecifically to lysine located at the 19th or 134th position in theamino acid sequence of interferon-β. An interferon-β complex produced bya method of the present invention maintains high activity, while havingsufficient solubility and physical and biological stability as well asexcellent circulatory half-life and clearance values, in living bodies.Thus, the interferon-β complex of the present invention produces fewerside effects and is useful as a highly effective pharmaceutical drug.

1. An interferon-β complex produced by binding interferon-β comprisingthe amino acid sequence of SEQ ID NO: 1, or a mutant thereof which hasantiviral activity and comprises a deletion of Met¹ and a substitutionof Cys¹⁷ with Ser in the amino acid sequence of SEQ ID NO: 1, whereinthe interferon-β or the mutant thereof may or may not contain sugarchains, to polyethylene glycol, which has an average molecular weight ofapproximately 40,000 Da or above, at a lysine located at the 19^(th) or134^(th) position of the interferon-β, or at a lysine located at aposition corresponding thereto of the mutant, in an amino acid sequenceof the interferon-β or the mutant thereof, in the presence of at leastone additive selected from the group consisting of disaccharides,monosaccharides, sugar alcohols thereof, and C₂₋₃ polyhydric alcohols,wherein the antiviral activity of the complex is at least 10% of theantiviral activity of interferon-β before binding to polyethyleneglycol.
 2. A pharmaceutical composition comprising aninterferon-βcomplex according to claim
 1. 3. The interferon-β complexaccording to claim 1, wherein the interferon-β is natural or recombinantinterferon.
 4. The interferon-β complex according to claim 1, whereinthe polyethylene glycol has an average molecular weight of approximately40,000 Da to 60,000 Da.
 5. The interferon-β complex according to claim1, wherein the additive is selected from the group consisting ofglucose, mannitol, sorbitol, sucrose, trehalose, ethylene glycol, andglycerol.
 6. An interferon-β complex of interferon-β and polyethyleneglycol, wherein the interferon-β is interferon-β comprising the aminoacid sequence of SEQ ID NO: 1, or a mutant thereof which has antiviralactivity and comprises a deletion of Met¹and a substitution of Cys¹⁷with Ser in the amino acid sequence of SEQ ID NO: 1, wherein theinterferon-β or the mutant thereof may or may not contain sugar chains,and wherein the polyethylene glycol, which has an average molecularweight of approximately 40,000 Da or above, is bound to a lysine locatedat the 19^(th) or 134^(th) position of the interferon-β, or a lysinelocated at a position corresponding thereto of the mutant, in the aminoacid sequence of the interferon-β or the mutant thereof, wherein theantiviral activity of the complex is at least 10% of the antiviralactivity of interferon-β before binding to polyethylene glycol.
 7. Theinterferon-β complex according to claim 6, wherein the polyethyleneglycol has an average molecular weight of approximately 40,000 Da to60,000 Da.
 8. A pharmaceutical composition comprising aninterferon-βcomplex according to claim
 6. 9. The interferon-β complexaccording to claim 6, wherein the interferon-β is natural or recombinantinterferon.